This invention pertains to the detection of carbohydrates, and to synthetic compounds that exhibit colorimetric or fluorimetric responses in the presence of sugars and other carbohydrates.
Several efficient methods are available for analyzing amino acids or nucleic acids. By contrast, no single method is available that is suitable for the quantitative or qualitative analysis of saccharides generally. The high degree of structural similarity between different sugars hinders their selective detection. The direct visible detection of sugars is especially challenging, since unmodified saccharides generally do not absorb light in the visible region. See generally M. Chaplin, xe2x80x9cMonosaccharides,xe2x80x9d pp. 1-41 in M. Chaplin et al. (Eds.), Carbohydrate Analysis. A Practical Approach (Oxford University Press 1994); and J. Kennedy et al., xe2x80x9cOligosaccharides,xe2x80x9d pp. 43-67 in M. Chaplin et al. (Eds.), Carbohydrate Analysis. A Practical Approach (Oxford University Press 1994).
Color assays for saccharides have been reported, including those based on certain synthetic molecules and those based on certain enzymes. Color assays based on synthetic molecules are typically less expensive than enzymatic methods, and their reagents are generally more resistant to degradation. Enzymatic assays can offer greater specificity than the non-enzymatic color tests, but they are generally more expensive, and their reagents are less stable. The inherently unstable enzymes must be protected from extreme conditions during manufacture, storage, and use. An ideal detection technique for sugars would be highly specific, and would employ relatively inexpensive and stable, non-enzymatic reagents.
Problem 1. Selective Visible Detection of Fructose.
Fructose is a nearly ubiquitous component of nutrient products. Non-enzymatic glycosidation products form more rapidly in vivo from fructose than from glucose. Fructose is absorbed by the gastrointestinal tract more slowly than is glucose, and does not require insulin for entry into the liver. These features make it appealing for use by diabetics. However, fructose has a higher tendency to be converted to fat rather than glycogen, thereby producing elevated blood triglyceride levels. High D-fructose intake has been implicated in the pathogenesis of hypertriglyceridaemia, atherosclerosis, and insulin resistance.
A glucose-fructose syrup is used in many food and beverage products. The worldwide production of high fructose syrup (HFS) is currently about 8xc3x97109 kg per year.
Carbohydrates comprise  greater than 98% of the soluble solids in fruit juices such as apple juice and orange juice. For example, fructose and glucose are the main carbohydrate constituents of apple juice, in a ratio greater than 2 fructose: 1 glucose.
There is an unfilled need for a simple and rapid color test that is highly specific for fructose, even in the presence of other monosaccharides such as glucose, a color test that does not require the corrosive, expensive, or degradable materials that are the basis of current monosaccharide color assays. Such a test could be of global benefit to industry and biomedicine.
The selective determination of fructose in plasma is especially challenging. Glucose is typically present in plasma in 100-fold excess of fructose. The determination of fructose levels in human plasma cannot currently be performed reliably, largely due to the xe2x80x9cexcessxe2x80x9d glucose levels. Measured levels of plasma fructose thus vary greatly among laboratories, and vary by the technique employed.
The AOAC (formerly the Association of Official Analytical Chemists) official methods for the analysis of fructose rely principally on gas chromatography (GC), and high performance liquid chromatography (HPLC) with refractive index detection. The gas chromatography analysis typically employs a prior derivatization of the sugars (e.g., methylation or trimethylsilation). Refractive index detection in HPLC is subject to significant cross-sensitivity to other, non-specific sugars and biomolecules. More recently, an electrochemical method, pulsed amperometric detection (PAD), has gained widespread use for monosaccharide detection in conjunction with HPLC; however, PAD is limited by the need to operate at high pH which, in turn, limits the choice of solvents and conditions. Mass detectors can be very useful when coupled to HPLC or GC systems; however, mass spectroscopy adds a further degree of complication and expense to the analysis. Simple reducing sugar assays can be used in automated post-column detection systems for monosaccharides including fructose; however, they also require harsh reagents and conditions. Enzyme-based assays have also been used for specific fructose determination; however, enzymes are expensive and are readily degradable.
Recent studies have described the visual color sensing of monosaccharides, including fructose, by boronic acid-appended dyes. These techniques rely on sensing changes in color promoted either by the perturbation of an aggregation-disaggregation equilibrium (i.e, the addition of saccharides promotes the disaggregation of the boronic acid-functionalized dye); or by the perturbation by a sugar of the interaction of the boronic acid with a neighboring amine (attached to an azo dye), producing charge transfer effects. See, e.g., T. James et al., xe2x80x9cSaccharide Sensing with Molecular Receptors Based on Boronic Acid,xe2x80x9d Angew. Chem. Int Ed. Engl., vol. 35, pp. 1910-1922 (1996); K. Koumoto et al., xe2x80x9cDesign of a Visualized Sugar Sensing System Utilizing a Boronic Acid-azopyridine Interaction,xe2x80x9d Supramolecular Chemistry, vol. 9, pp. 203 ff(1998); and K. Koumoto et al., xe2x80x9cColorimetric Sugar Sensing Method Useful in xe2x80x98Neutralxe2x80x99 Aqueous Media,xe2x80x9d Chem. Lett., pp. 856-857 (2000).
In one modification, an optical wavelength shift has been observed following the binding of glucose in aqueous methanol; however, this modified system operates only at high pH ( greater than 12). See C. Ward et al., xe2x80x9cA Molecular Colour Sensor for Monosaccharides,xe2x80x9d J. Chem. Soc., Chem. Commun., pp. 229-230 (2000).
Our research group has previously reported certain boronic acid-containing resorcinol condensation products (compounds 1 and 2 below), and their use in the non-selective color detection of sugars. See P. Lewis et al., xe2x80x9cTetraarylboronic Acid Resorcinarene Stereoisomers. Versatile New Substrates for Divergent Polyfunctionalization and Molecular Recognition,xe2x80x9d J. Org. Chem., vol. 62, pp. 6110-6111 (1997); and C. Davis et al., xe2x80x9cSimple and Rapid Visual Sensing of Saccharides,xe2x80x9d Organic Letters, vol. 1, pp. 331-334 (1999). Although different colors were observed for reactions with different sugars in the latter paper, the method of this paper is considered non-selective in the sense that it does not allow the selective detection of a single sugar of interest when it occurs in a background of other sugars. 
Problem 2. Mild and Selective Detection of Sialic Acid.
Sialic acids are common components of glycoproteins, glycopeptides and glycolipids. Sialic acids play a role in cell-to-cell communication in humans and other animals, and have been implicated in increased virulence in some bacteria. Imbalances in sialic acid levels can alter cell adhesion, which may have an effect in certain cancers and in some types of graft rejection. An increase in either soluble or cellular sialic acid levels can be a diagnostic marker for cancer. The function of sialic acids is incompletely understood. They appear to act as amphiphilic donors of negative charge to the cell surface. Improved methods for analyzing sialic acids would greatly aid in the elucidation of their biochemistry and in the detection of certain cancers.
The most commonly used assays for sialic acid are probably the Warren assay and the Svennerholm color test. These assays require high temperatures, harsh and toxic reagents, and are subject to interference from other carbohydrates. Free sialic acids for the Warren and Svennerholm assay are obtained by either acidic or enzymatic (e.g., neuraminidase-promoted) hydrolysis. Acidic hydrolysis leads to the liberation of both sialic acids and L-fucose. Unfortunately, L-fucose interferes with the absorbance for sialic acid measured in the Warren assay. Additionally, fructose interferes with both the Warren and Svennerholm assays. See, e.g., I. Sobenin et al., xe2x80x9cOptimization of the assay for sialic acid determination in low density lipoprotein,xe2x80x9d J. Lipid Res., vol. 39, pp. 2293-2299 (1998); and R. Mattoo et al., xe2x80x9cQuantitative determination of sialic acid in the monosialoganglioside, GM1, by the thiobarbituric acid method,xe2x80x9d Anal. Biochem., vol. 246, pp. 30-33 (1997).
The Warren assay typically uses a harsh periodate oxidation of sialic acid, treatment with phosphoric acid, treatment with toxic sodium arsenite, and the use of corrosive sulfuric acid. Also required are further treatment with thiobarbituric acid and redistilled cyclohexanone, and 15 min heating at 100xc2x0 C.
There is an unfilled need for an assay for sialic acid that does not require harsh reagents or harsh conditions, and that is not subject to substantial interference from fructose, fucose, and other neutral carbohydrates.
Problem 3. Color Detection of Oligosaccharides.
Glycobiology has attracted much recent attention due, in large part, to the therapeutic potential of many oligosaccharides. Many different oligosaccharides naturally occur in glycoproteins and in cell surfaces. The problems of analyzing monosaccharides are compounded with oligosaccharides due to the tremendous variety of linear and branched oligosaccharides.
A leading author has observed, xe2x80x9cDetection of oligosaccharides eluting from HPLC columns is the biggest challenge and weakest link in the analysis of oligosaccharides.xe2x80x9d J. Kennedy et al. (1994), p. 62. There is an unfilled need for a simple method for the visible detection of oligosaccharides. There are currently no useful direct color tests for higher molecular weight oligosaccharides. Refractive index detectors are typically used to detect oligosaccharides in HPLC analyses, but refractive index detection is highly sensitive to the temperature used and the nature of the mobile phase employed, and refractive index detection is also susceptible to non-specific cross-reactivity. UV detection at wavelengths below 210 nm is another option, but UV detection limits solvent choice and requires expensive ultrapure solvents to reduce interferences. Electrochemical detection by pulsed amperometric detection (PAD) requires high pH conditions. Mass spectrometry, coupled with chromatographic separations, requires highly specialized and expensive equipment. Aromatic or heterocyclic substituents have been used as chromogenic labels to facilitate UV detection of oligosaccharides. Radioactive labeling has also been used, but has the obvious disadvantage that it requires the handling of radioactive substances.
Classical color tests for monosaccharides fail to reliably detect oligosaccharides containing more than three monosaccharide residues. The color response in a classical color test is typically a function of the molar concentration of oligosaccharide, not its concentration by weight. For example, the response of maltohexaose has been reported to be about 18% of that for the same concentration by weight of glucose (maltohexaose is comprised of six glucose units). J. Kennedy et al. (1994), p. 46.
We have discovered novel methods for the simple, rapid, and selective colorimetric detection of carbohydrates, including fructose, glucose, sialic acid, and oligosaccharides. There is no need for any prior hydrolysis or other chemical modification or of the analytes. Resorcinarenes, xanthene dyes, and related compounds, formally produced by the reaction of 2 equivalents of resorcinol and a suitable electrophilic condensation partner, are used as chromophores or fluorophores for the detection of sugars and other carbohydrates.
The receptors of the novel method require neither azo moieties nor amine moieties, and they do not tend to aggregate in solution. High pH is not required.
Fields in which the present invention should prove useful include medical diagnostics, quality control, the fermentation industry, breweries, the food industry. in all these fields, the detection of saccharides is of great importance.
The novel method for the detection of fructose provides a simple, rapid procedure using relatively inexpensive and stable synthetic reagents. The method affords the highest selectivity of any known synthetic receptor for fructose in the presence of glucose at room temperature. For example, in the presence of 100 equivalents of glucose the absorbance of the novel chemosensor/fructose complex at 464 nm has been seen to be virtually unchanged as compared to that of fructose alone (in the absence of glucose). By contrast, absorbance at 535 nm changes as a function of glucose concentration. Thus monitoring absorbance at two wavelengths allows for the simultaneous determination of both fructose and glucose levels. We have observed that the method is also selective for fructose in the presence of other neutral carbohydrates, for example, xcex1-lactose monohydrate, maltose monohydrate, D-(+)-xylose, D-(+)-glucose, and sucrose.
No synthetic receptor for detecting fructose at visual wavelengths has been previously reported that is not subject to significant interference from other commonly occurring carbohydrates, such as glucose. The current invention achieves an unprecedented degree of selectivity for fructose using a method that is easy to implement, and that does not require harsh reaction conditions.
The novel method for detecting sialic acid was not adversely affected by the presence of other carbohydrates, for example, D-(xe2x88x92)-fructose, L-fucose, D-(+)-glucose, and D-glucosamine hydrochloride; nor was the method adversely affected by the presence of amino acids, for example, L-cysteine. The sialic acid analysis is fast and simple, and can be successfully used at mild, room-temperature conditions using non-corrosive reagents at neutral or near-neutral pH.
In the presence of compounds 4 and 5, no significant relative decrease in visible absorbance intensities were observed for glucose and the maltodextrin oligomers, a prototypical series of carbohydrates. No prior hydrolysis or chemical modification of the sugars with a chromogenic reagent was required. Neutral oligosaccharides of progressively larger size were thus directly detectable with our method. The structures of compounds 4 and 5 are depicted below. 
The novel color test for detecting oligosaccharides is simple to administer. In prototype studies, we have examined typical neutral oligomers composed entirely of glucose monomers, the malto-oligosaccharides. The malto-oligosaccharides (maltose through maltohexose), each at the same concentration of 4 mg in 2 mL, in the presence of compound 1, exhibited unprecedented absorbance responses in the visible region. The structure of compound 1 is depicted below: 
The novel methods use resorcinol condensation products (e.g., compounds 3, 4 and 6) or commercially available xanthene chromophores (e.g., compound 5) as colorimetric or fluorimetric sugar detection agents. Xanthene dyes (which include fluorescein, rose bengal, ethyl eosin, and others) are of great importance in photochemistry, photomedicine, photographic technology, tunable lasers, and fluorescence depolarization diagnostic devices. They have been used both as electron acceptors and as electron donors (to initiate free radical polymerizations), depending on the reactant. To the inventors"" knowledge, there have been no prior reports of using xanthene compounds in saccharide-induced color changes. The structures of compounds 3, 4, 5 and 6 are depicted below: 
We have achieved several advances over the results previously reported by our research group and by others. We have now achieved the selective colorimetric detection of monosaccharides, at room temperature under neutral or near-neutral (preferably buffered) conditions. Color or fluorescence detection using compounds 3-6 is believed to be novel. Sugar color detection using resorcinarene or xanthene condensation products without boron has not been previously reported. Useful oligosaccharide color detection using mild reaction conditions is itself new. The new methods greatly simplify the color detection of mono- and oligosaccharides, without the need for corrosive reagents.
The compounds used in this invention generally are soluble in a water/aprotic polar solvent mixture, for example a mixed water/dimethylsulfoxide (DMSO) solvent. Other aprotic polar solvents that may be used such a mixture are well known in the art, and include, for example, tetrahydrofuran, dimethylformamide, sulfolane, acetonitrile, and hexamethylphosphoramide.